Oncogene (2002) 21, 6209 – 6221
ª 2002 Nature Publishing Group All rights reserved 0950 – 9232/02 $25.00
www.nature.com/onc
Control of the centriole and centrosome cycles by ubiquitination enzymes
David V Hansen1,2, Jerry Y Hsu1,2, Brett K Kaiser1,2, Peter K Jackson*,1,2 and Adam G Eldridge1,2
1
Programs in Chemical Biology and Cancer Biology, Stanford University School of Medicine, 300 Pasteur Drive, Stanford,
California, CA 94305-5324, USA; 2Department of Pathology, Stanford University School of Medicine, 300 Pasteur Drive,
Stanford, California, CA 94305-5324, USA
Oncogene (2002) 21, 6209 – 6221. doi:10.1038/sj.onc.
1205824
Keywords: ubiquitin ligases; ubiquitination; centrosome
duplication
Introduction
The role of the centriole in organizing the cell’s
cytoskeleton and its mechanism of duplication have
been long-standing puzzles for cell biologists. Whereas
the semi-conservative replication of chromosomes was
established by Meselson and Stahl (1958), the likely
parallels for semi-conservative duplication of the
centrioles remain fuzzy. Further considering this
parallel, molecular studies of chromosomal replication
have begun to uncover how cell cycle regulators
including cyclin-dependent kinases and ubiquitin ligases
ensure that chromosomes replicate once-and-only-once
per cell cycle (Blow and Hodgson, 2002; Dutta and Bell,
1997). The obvious need to maintain accurate control of
centrosome number and thereby ensure spindle bipolarity would suggest that a similar once-and-only once
control restricts the centrosome cycle.
Studies over the last decade on the budding and
fission yeast spindle pole bodies (SPB) and the animal
cell centrosome have defined a growing parts list of
conserved components, as well as those specific to
fungi or animals. The functional connection between
the centrosome duplication cycle and the regulatory
mechanisms controlling the chromosome duplication
cycle suggested that semi-conservative replication for
both centrosomes and chromosomes might be linked
by these global timing mechanisms. In 1999, a series
of studies demonstrated that cyclin-dependent kinases
and both the SCF and APC ubiquitin ligases – cell
cycle regulators already well established in control of
the chromosome replication cycle – also had fundamental roles in controlling the centrosome cycle
(Freed et al., 1999; Hinchcliffe et al., 1999; Lacey et
al., 1999; Meraldi et al., 1999; Vidwans et al., 1999).
Since then, a number of other cell cycle regulators
have been directly implicated in the centrosome cycle,
*Correspondence: PK Jackson; E-mail: [email protected]
many of which are described in the accompanying
reviews. Here we will focus on the role of ubiquitin
ligases in controlling the centrosome cycle, considering
both those known or postulated core centrosomal
factors that are directly ubiquitinated, as well as
ubiquitination of specific cell cycle regulators – including kinases and ubiquitin ligases themselves – that
more globally control the centrosome cycle. First, we
will review the biochemistry of ubiquitin ligases, and
then return to the role of these enzymes in the various
phases of the centrosome cycle.
The biochemistry of ubiquitin ligases
The addition of polymeric chains of ubiquitin to lysine
side chain residues of specific proteins is a sufficient
signal to target the proteins to the 26S proteasome for
proteolytic destruction. Ubiquitination of a substrate
requires a ubiquitin enzyme shuttle using E1, E2, and
E3 enzymes to direct the formation of ubiquitin chains
on specific substrates. Among these ubiquitination
enzymes, the E3 ubiquitin ligases appear to provide
the critical elements of specificity that direct the
formation of polyubiquitin chains on protein targets
(See Appendix). Ubiquitin-dependent proteolysis is a
widely used mechanism for regulating protein function
in processes ranging from cell cycle and developmental
switches to homeostatic control of environmental
sensing (see reviews: Deshaies, 1999; Jackson et al.,
2000).
Briefly, after activation by an E1 enzyme, the E2
ubiquitin-conjugating enzyme and E3 ubiquitin ligase
cooperate to assemble the polyubiquitin chain on a
protein substrate. In studies so far, the E3 ubiquitin
ligase uses protein-protein interaction domains outside
the catalytic domain to bind substrate (Figure 1). To
date, there are two major structural classes of E3
ubiquitin ligases, although newer studies suggest
additional domains may possess E3 activity (Hatakeyama et al., 2001; Jiang et al., 2001; Lu et al., 2002;
Patterson, 2002). Of the two major classes, the HECT
domain proteins are a modest sized family of proteins
with no currently known connections to centrosome
biology. They have been recently reviewed (Jackson et
al., 2000) and will not be addressed further here. In the
largest class of E3 ubiquitin ligases, the so-called
RING (for ‘Really Interesting New Gene’) finger
Ubiquitination and the centrosome cycle
DV Hansen et al
6210
Figure 1 A summary of RING finger ubiquitin ligases. Presented are representative examples of RING finger ubiquitin ligases
discussed in the text
proteins use a zinc-binding function to stimulate
catalysis of ubiquitin chain formation and may
combine substrate binding and catalytic domains
within a single polypeptide. Alternatively, a smaller
RING finger polypeptide can be associated within the
context of a multi-protein complex with separate
subunits containing (i) the RING finger and associated
catalytic polypeptides; and (ii) substrate binding (or
‘adapter’) functions.
SCF (Skp1, Cullin, F-box) complexes
The SCF class of ubiquitin ligases contains at least
four proteins: Skp1, the cullin protein Cul1, the Ring
Finger protein Rbx1, and an F-box protein, a substrate
binding protein (see Figure 1). Other proteins
associated with SCF ubiquitin ligases are still being
defined, but biochemical reconstitution of these four
components plus E1 and E2 enzymes is sufficient for
ubiquitin chain formation on an appropriately modified substrate.
SCF substrates are bound directly by the F-box
proteins, which contain a *45 amino acid motif called
an F-box and bind to substrates through proteinprotein interaction domains (Jackson et al., 2000). The
F-box is required for binding to Skp1, which in turn
associates with Cul1. The cullin protein acts as a
scaffold, bringing the E2 ubiquitin-conjugating enzyme
into close proximity with the rest of the complex
Oncogene
(Zheng et al., 2002). There are at least six human
cullins, including Cul1-5 and Apc2. Apc2 functions in a
known E3 complex (discussed below). The remaining
cullins are likely to organize ubiquitin ligase complexes
similar to SCF and Cul1. Human Roc1/Rbx1/Hrt1, a
protein containing a RING-H2 finger domain, appears
to promote association of the cullin protein with the
E2 enzyme and to enhance ubiquitin ligase activity
(Kamura et al., 1999; Ohta et al., 1999; Skowyra et al.,
1999; Tan et al., 1999). The RING-H2 is one of a class
of RING finger proteins containing an octet of cysteine
and histidine residues that participate in E2 binding
and catalysis.
The F-box adapter proteins direct ubiquitination of
diverse substrates. There are 16 F-box proteins in S.
cerevisiae, *100 in the complete C. elegans genome,
and more than 50 described so far in vertebrates
(Jackson et al., 2000). In the highly studied SCFCdc4,
SCFSkp2 and SCFb7TrCP complexes, substrate phosphorylation is required for binding the F-box protein.
In the budding yeast SCFCdc4 complex, a WD40repeat protein, Cdc4, binds each of the known
substrates (Sic1p, Cdc6p, Gcn4p) in a phosphorylation-dependent fashion. The SCFb7TrCP recognizes a
specific phosphoserine motif (DSGfXS) found in the
regulatory proteins IkBa and b-catenin. Skp2, a
leucine-rich repeat F-box protein, also recognizes only
the phosphorylated form of the Cdk regulator p27Kip1.
Although phosphorylation is clearly required for the
Ubiquitination and the centrosome cycle
DV Hansen et al
6211
binding of specific substrates to their F-box adapters,
it is not clear whether phosphorylation merely serves
to improve the affinity of substrate to F-box, or has
another role. In part, the insolubility of several F-box
proteins has limited these types of biochemical studies.
The anaphase promoting complex (APC) ubiquitin ligase
The APC was the first multicomponent ubiquitin ligase
described and is required for the degradation of
substrates controlling the metaphase-to-anaphase transition and for the destruction of cyclin B to allow exit
from mitosis. Similar to the SCF complex, the APC
contains a cullin homolog, Apc2, and a RING-H2
finger protein similar to Rbx1, called Apc11. Reconstitution studies show that these two proteins form the
catalytic core of the APC and are sufficient for
ubiquitin chain assembly in vitro (Gmachl et al.,
2000; Tang et al., 2001).
The APC associates with two WD repeat-containing
adapter proteins, Cdc20/Fizzy and Cdh1/Hct1/Fizzyrelated. Both bind the APC and have recently been
shown to be important for substrate binding (Burton
and Solomon, 2000; Hilioti et al., 2001; Pfleger et al.,
2001a; Schwab et al., 2001), although the specific
determinants of binding remain unclear. The APC in
humans and yeast also contains other components (at
least 11 polypeptides in total), although the role of
these additional components and the possible heterogeneous composition of the APC are not understood.
Two general destruction signals have been identified in
substrates targeted for destruction by the APC, the
destruction box (or D-box) and the KEN box (Pfleger
and Kirschner, 2000). The 9 amino acid D-box is
found in all the known APCCdc20 substrates, but also
in some APCCdh1 substrates. In addition, the KEN
box was described as a transposable, 7 amino acid
motif that appears to target substrates specifically to
APCCdh1. Thus, Cdh1 can target ubiquitination of
both D-box and KEN box containing substrates,
including Cdc20 itself. Although the D-box and
KEN box motifs appear to be necessary for Cdc20and Cdh1-activated substrate ubiquitination, it is not
clear whether Cdc20 or Cdh1 are sufficient for direct
binding of these recognition sequences or whether
additional sequences or factors can contribute to
adapter binding.
Ubiquitination of APC substrates is regulated both
by phosphorylation of the APC core components and
by phosphorylation of at least the Cdh1 adapter
protein (Kramer et al., 2000). The APC is also
controlled by inhibitory proteins that bind to the
Cdc20 and Cdh1 adapter proteins. These include the
spindle assembly checkpoint protein Mad2, which
binds Cdc20 and inhibits APC activity following
spindle damage (Shah and Cleveland, 2000), a closely
related protein, called Mad2B, which binds and inhibits
Cdh1 (Chen and Fang, 2001; Pfleger et al., 2001b), and
the Emi1 protein (Hsu et al., 2002; Reimann et al.,
2001a,b), which binds both Cdc20 and Cdh1 to inhibit
their activity during interphase.
Single polypeptide RING finger E3 proteins
A number of E3 ubiquitin ligases have been found to
contain an N-terminal substrate binding domain and a
C-terminal RING finger within the same polypeptide.
In one of the better-studied examples, the p53
regulator Mdm2 uses an N-terminal p53-binding
function and a C-terminal RING-H2 finger to direct
the ubiquitination of the tumor suppressor p53
(Honda et al., 1997).
An overview of the events of centriole and centrosome
duplication throughout the cell cycle
As cells exit mitosis and begin cell growth during G1
phase, each daughter cell has received one centrosome
containing a paired centriole (or ‘duplosome’), which
serves as the microtubule organizing center (MTOC)
for the cell (Figure 2). The centriole appears to
organize the pericentriolar material (PCM), including
a ‘centromatrix’ and many copies of the gamma
tubulin ring complex (g-TuRC), which nucleates
microtubule outgrowth. In many animal cells, these
centrioles split around the time of the G1/S transition,
when DNA replication begins, but this is by no means
universal (Vidwans et al., 1999). Upon S phase entry,
the two separated centrioles (‘mother’ centrioles) each
nucleate the outgrowth of a procentriole (‘daughter’),
the elongation of which occurs during S and G2 phases
and is completed in mitosis. Because the original
centrosome was composed of a mother-daughter
centriole pair, one of the split centrioles is an older
mother and the other a younger or new mother.
During G1 phase, the centriolar pair has MTOC
activity, which is thought to be generated by the older
mother centriole (Hinchcliffe and Sluder, 2001). After
centriolar splitting and the outgrowth of procentrioles
from the older and new mothers, the new mother must
acquire MTOC activity as a prerequisite to forming the
other pole of the mitotic spindle. This functional
maturation late in S/G2 phase is correlated with the
formation of the distal appendages, which emanate
from the elongating mother centriole (Bornens, 2002;
Marshall, 2001), and appear in immunoelectron
micrographs as the site of microtubule outgrowth
(Paintrand et al., 1992).
Just before mitotic entry, the elongation of the
procentrioles is nearly completed and the two
centriolar pairs organize a more elongated PCM. The
centriolar pairs appear to be tethered by a fibrous
bridge (Paintrand et al., 1992), which also contains
SCF components including the protein Skp1 (Freed et
al., 1999). Near the time of mitotic entry, the
centrosome splits – possibly by dissolution of this
bridge – and the PCM organized around each
centriole increases dramatically in size (Khodjakov
and Rieder, 1999). This increase is not dependent on
microtubules, but possibly on the activation of mitotic
kinases. These coupled events provide the mechanism
to build the bipolar mitotic spindle.
Oncogene
Ubiquitination and the centrosome cycle
DV Hansen et al
6212
Figure 2 A schematic of the centriole and centrosome cycles
Following spindle assembly and the triggering of
chromosome segregation, a set of chromosomes and
one centrosome are pulled toward each pole of the
elongating cell and cytokinesis is triggered between the
spindle poles to ensure that each daughter cell receives
both a full complement of chromosomes and a single
centrosome (Glotzer, 2001). Recent analysis from the
Bornens’ lab suggests that the centrioles are considerably more dynamic than previously thought and that
the mother centriole plays a critical role in triggering
cytokinesis (Piel et al., 2000, 2001). Thus, our current
model for the centriole and centrosome cycle may
require some fundamental revision.
The specific order of morphological events in the
centrosome cycle suggests an orderly pathway. The
close linkage of centrosomal events in step with cell
cycle transitions has suggested the coupling of cell cycle
regulators including ubiquitin ligases and cyclindependent kinases to the centrosome cycle. We will
now examine the role of these ubiquitin ligases in
specific transitions within the centrosome cycle.
Ubiquitin-dependent proteolysis in centriole separation
The first step of the centrosome duplication cycle
begins with centriolar splitting at the G1/S transition,
thereby allowing subsequent procentriolar outgrowth.
The cyclin-dependent kinase Cdk2 has been shown to
be important for centrosome duplication. Specifically,
cyclin E/Cdk2 activity is necessary for centrosome
duplication in Xenopus embryos and centriole separation in extracts from these embryos (Lacey et al., 1999;
Oncogene
Hinchcliffe et al., 1999). Similar requirements for Cdk2
activity in centrosome duplication have been shown in
somatic cells (Matsumoto et al., 1999), although cyclin
A/Cdk2 instead of cyclin E/Cdk2 may be the cyclindependent kinase necessary for centrosomal duplication in somatic cells (Meraldi et al., 1999).
The Cdk2 activity necessary for centriole separation
may first play a positive role in phosphorylating some
factor necessary for centriole splitting, similar to a role
it is thought to play in triggering DNA replication
(Dutta and Bell, 1997). Additionally, Cdk2 may
phosphorylate and inactivate an inhibitory protein,
possibly by triggering its destruction. By analogy, the
Cdk2 kinase triggers destruction of SCF substrates like
the cyclin E/Cdk2 inhibitor p27Kip1 to promote the
G1/S transition. At this point, we have only one
candidate for the cyclin E phosphorylation target at
the centrosome, nucleophosmin (Okuda et al., 2000),
but additional studies will be necessary to better
understand the implications of this work. It does
appear that there must be targets that are not simply
Cdk2 inhibitors (discussed below). In an interesting
twist, the kinase Mps1 is a positive regulator of
centrosome duplication and an in vitro substrate of
Cdk2. Inhibition of Cdk2-dependent phosphorylation
results in proteasome-dependent degradation of murine
Mps1 (Fisk and Winey, 2001). Thus, it is possible that
the Mps1 dephosphorylation allows its recognition by
a yet unidentified E3 ubiquitin ligase.
A specific role for SCF ubiquitin ligases in the
centrosome duplication cycle was suggested by the
discovery that the proteins Skp1 and Cul1, two core
components of the SCF ubiquitin ligase, localize to the
Ubiquitination and the centrosome cycle
DV Hansen et al
6213
centrosome by indirect immunofluorescence and immunoelectron microscopy (Freed et al., 1999; Gstaiger et
al., 1999). These proteins were also highly enriched in
purified centrosomes and the centrosome-localized
cullin Cul1 was quantitatively modified by Nedd8, a
ubiquitin-like molecule necessary for SCF ubiquitination activity. This enrichment of Nedd8-modified Cul1
at the centrosome suggested that the centrosomally
localized SCF complex was highly active. Centriole
separation was inhibited by neutralizing antibodies to
Skp1 or Cul1 in Xenopus extracts and could not be
restored by addition of recombinant cyclin E/Cdk2
(Freed et al., 1999), suggesting that SCF-mediated
proteolysis of a target other than a cyclin-dependent
kinase inhibitor is required for centriole separation.
Inhibition of the proteasome by addition of the
proteasome inhibitor MG132 also resulted in inhibition
of centriole separation in vitro and prevented centrosome duplication when injected into Xenopus embryos,
further suggesting a role for proteolysis at this stage of
the centrosome cycle.
The Anaphase Promoting Complex (APC) also has a
role in controlling events at the G1/S transition,
notably by inhibiting the accumulation of cyclin A,
which as mentioned before is important for triggering
centrosome duplication in somatic cells (Meraldi et al.,
1999). Thus, the ability of the APC to restrain cyclin A
accumulation may be important in preventing premature centriole separation. Recent studies show that the
human Emi1 protein can inhibit the APCCdh1 complex
and thereby stabilize cyclin A to drive entry into S
phase (Hsu et al., 2002). Perhaps Emi1 also participates
in the regulation of centriole splitting at the G1/S
transition.
Limiting centrosome duplication to once per cell cycle
The original cell fusion studies of Rao and Johnson
(1970) provided evidence not only for S phase
promoting factor and M-phase promoting factor, but
also for a mechanism to limit rereplication of a G2
nucleus until cells had passed through mitosis. The
mechanism for limiting DNA replication to once-andonly-once per cell cycle is controlled by at least two
mechanisms.
First,
replication
pre-inititiation
complexes form only when Cdk activity is low in G1
phase, and second, the activation of DNA unwinding
and polymerase functions occurs when Cdk activity is
raised at the G1/S transition (Blow and Hodgson,
2002; Dutta and Bell, 1997). At the same time, the high
Cdk activity required to trigger initiation inhibits the
reformation or reuse of preinitiation complexes, thereby ensuring that triggering of replication initiation is
linked to preventing the establishment of preinitiation
structures. The process of reestablishing pre-initiation
complexes is reset during mitosis by a mechanism that
requires the destruction of the mitotic cyclins by the
APC (Noton and Diffley, 2000). However, the block to
rereplication can be lost by inactivating Cdk activity
during S or G2 phases, which allows premature
assembly of replication preinitiation structures and a
round of rereplication without an intervening mitosis.
Given the parallels between the triggering of DNA
replication and centrosome duplication by Cdks, it is
tempting to speculate that a similar once-and-only-once
mechanism regulates centriole duplication.
A surprising aspect of the centrosome cycle is that
treatments that arrest cells in S phase allow an eventual
uncoupling of the centrosome cycle from the mitotic
cycle, giving rise to ectopic accumulation of centrosomes. Brinkley and colleagues first demonstrated that
arresting Chinese Hamster Ovary (CHO) cells in S
phase with hydroxyurea blocked DNA replication, but
resulted in overduplication of centrosomes with some
cells showing 8, 16, or more centrosomes (Balczon et
al., 1995). A similar experiment in Xenopus embryos
demonstrated that arresting cells in interphase with the
protein synthesis inhibitor cycloheximide blocked
mitosis (by preventing accumulation of cyclin B), but
allowed the eventual overduplication of centrosomes
(Gard et al., 1990).
Although it is not clear what allows the uncoupling
of the centrosome cycle, the ability of the centrosome
in the S phase-blocked cell to resume Cdk and SCFdependent duplication appears to be an adaption that
occurs following a prolonged S phase block. In CHO
cells arrested with hydroxyurea, the cells do not begin
centrosome overduplication for a period longer than
the time required for a full cell cycle. Thus, it may be
that the normal mechanisms gating centrosome division are lost over time and ectopic centrosome
duplication ensues. For example, if the activity of the
APC normally caused the rapid destruction of some
unstable inhibitor in mitosis, this could be the normal
means to reset the centrosome and allow new
duplication. However, if during a prolonged S phase
arrest the slow destruction of this inhibitor occurred,
then the process of centrosome duplication could be
reinitiated. More detailed studies of the reduplication
phenomenon will be necessary to establish its mechanisms.
Centrosome reduplication would be deleterious to
the genomic stability of the cell by causing multipolar
spindles and thereby inducing chromosome segregation
defects. Such defects could also arise through abortive
cytokinesis following centrosome duplication, as has
recently been emphasized by Nigg and colleagues
(Meraldi et al., 2002). Here, the failure to segregate
centrosomes into daughter cells would cause an
increased centrosome number. The implications of
these kinds of defects for carcinogenesis are discussed
below.
Control of early mitotic events by ubiquitin-dependent
proteolysis
Several important steps in the centrosome duplication
cycle occur near the G2/M transition as mitotic cyclindependent kinase activity increases. These include the
completion of procentriole elongation, splitting and
Oncogene
Ubiquitination and the centrosome cycle
DV Hansen et al
6214
separation of the duplicated centriole pairs, and
functional maturation of the duplicated centrosomes.
Functional maturation is observed as a dramatic
increase in microtubule-nucleating capacity and concomitant recruitment of additional proteins to the PCM
(Khodjakov and Rieder, 1999). Although ubiquitinmediated proteolysis has not been directly implicated in
these processes, several key mediators of these events
are recognized for ubiquitination by the anaphasepromoting complex (APC) through its substrate
adapters, Cdc20 and Cdh1, and thus may be under
control of the Mad2 and Emil APC regulators. Thus,
controlled activation of the APC may directly or at
least indirectly prevent mitotic centrosome misfunction.
Centrosomal splitting
In mammalian cells, the centrosomal kinase Nek2A has
been shown to mediate the disjunction, or splitting, of
the duplicated centriole pairs through phosphorylation
of C-Nap1, which is believed to be an important
component of a proteinacious structure tethering the
proximal ends of the two parental centrioles (Fry et al.,
1998a,b; Mayor et al., 2000). Nek2A and protein
phosphatase type 1 (PP1) form a complex wherein each
enzyme is thought to antagonize the other’s activity
(Helps et al., 2000). The timing of centrosome splitting
around the time of mitotic entry may be attributed to
phosphorylation and inactivation of PP1 by cyclin B/
Cdc2 (Kwon et al., 1997), thus allowing phosphorylation of C-Nap1 by Nek2 to prevail over the opposing
phosphatase activity. C-Nap1 disappears from the
centrosome during prophase and reappears during late
telophase. The mechanism of how parental centrioles
disengage as a result of C-Nap1 phosphorylation is not
understood, but given that cellular levels of C-Nap1
decrease during mitosis (Mayor et al., 2000), an
intriguing possibility is that phosphorylation of CNap1 facilitates its recognition by a ubiquitin ligase
and that proteolysis is involved in the disassembly of
the tether. It is also possible that C-Nap1 phosphorylation is entirely sufficient for tether disassembly.
Recently, Nek2A was shown to be a substrate for
APCCdc20 with remarkable similarities to cyclin A – an
extended destruction box sequence mediating recognition by Cdc20, accumulation during S and G2 phases,
and proteolysis in early mitosis in the presence of an
activated spindle assembly checkpoint (Hames et al.,
2001). Nek2A has also been characterized as a
substrate for APCCdh1 ubiquitination through its
KEN-box motif (Pfleger and Kirschner, 2000). Given
the similarities between Nek2A and cyclin A, we might
expect that Nek2A accumulation would be regulated
by Emil (Hsu et al., 2002).
Centrosomal separation
The Eg5 family of kinesin-like proteins is important for
driving centrosome separation and properly maintaining spindle bipolarity (reviewed in Kashina et al.,
1997). In vertebrate tissue culture cells, the localization
Oncogene
of Eg5 to the mitotic spindle begins at the centrosomes
during G2 or prophase just prior to centrosome
separation and is dependent upon its phosphorylation
by cyclin B/Cdc2 (Blangy et al., 1995; Giet et al.,
1999a; Sawin and Mitchison, 1995). Also essential for
centrosome separation in higher eukaryotes are kinases
of the Aurora-A family, which, like Nek2A, are
antagonized by PP1 activity (Giet et al., 1999b; Glover
et al., 1995; Katayama et al., 2001). Aurora-A and Eg5
apparently act through the same pathway to drive
centrosome separation – Aurora-A phosphorylates and
co-immunoprecipitates Eg5, the two proteins colocalize from prophase through anaphase, and perturbing the function of either protein prevents the
bipolarity of spindles formed in Xenopus extracts (Giet
et al., 1999a; Roghi et al., 1998; Sawin et al., 1992).
Both Aurora-A and Cin8p, the budding yeast
homologue of Eg5, are degraded postmitotically. They
have recently been characterized as substrates for
ubiquitination by APCCdh1, and the amino acid motifs
required for their ubiquitination are highly conserved
among species (Arlot-Bonnemains et al., 2001; Castro
et al., 2002; Hildebrandt and Hoyt, 2001; Taguchi et
al., 2002). The vertebrate Eg5 has not been shown to
be regulated at the level of abundance, but it is possible
that critical pools of Eg5, such as those associated with
the centrosome, may be controlled by destruction.
Mitotic maturation of MTOC activity
Recruitment of additional proteins, including the gtubulin ring complex (g-TuRC), to the PCM and
enhancement of microtubule-nucleating capacity are
hallmarks of the mitotic centrosome. The accumulation
of the g-TuRC at the centrosome in mitosis is perturbed
by disrupting the function of Polo-like kinase 1 (Plk1),
Aurora-A, or Nek2B (Berdnik and Knoblich, 2002; do
Carmo Avides et al., 2001; Fry et al., 2000; Hannak et
al., 2001; Lane and Nigg, 1996; Uto and Sagata, 2000).
Like Aurora-A, the Polo-like kinase Plk1 has been
shown to be a substrate for post-mitotic APCCdh1mediated degradation in multiple species (Charles et al.,
1998; Fang et al., 1998; Shirayama et al., 1998). Whether
APCCdh1 also mediates the degradation of Nek2B is not
known; however, Nek2B protein levels oscillate in a cell
cycle-dependent manner reminiscent of other APCCdh1
substrates, being lowest in G1 and peaking in mitosis
(Hames and Fry, 2002).
A potential role for ubiquitin ligases in preventing mitotic
centrosome events in response to stress
The existence of checkpoints which arrest or delay
mitotic progression in response to various types of
stress improves the fidelity of chromosome segregation.
Arrest in G2 prevents mitotic entry by inhibiting Cdc2
activity through modulation of critical regulators of
Cdc2 (reviewed in OConnell et al., 2000), including the
phosphatase Cdc25C. APCCdh1 has recently been
reported to be an important component of the ionizing
radiation-induced G2 checkpoint (Sudo et al., 2001).
Ubiquitination and the centrosome cycle
DV Hansen et al
6215
Interestingly, the APCCdh1-mediated delay of mitotic
entry does not involve degradation of mitotic cyclins,
implying that the degradation of other APC targets
prevents mitotic entry. Supporting this idea, the G2/M
arrest induced by arsenite has been shown to include
ubiquitin-mediated degradation of Cdc25C in a KENbox dependent manner, suggesting that its ubiquitination is performed by APCCdh1 (Chen et al., 2002). In
Cdc25-null Drosophila embryos, which arrest in G2 of
cycle 14, procentriole elongation is attenuated, though
perhaps indirectly (Vidwans et al., 1999). Since the
mitotic activation of Nek2A, Eg5, and Aurora-A all
depend on Cdc2 activity, degradation of Cdc25C
would be predicted to prevent centrosomal splitting,
separation, and the acquisition of MTOC activity as
well. Alternatively, the APC could directly prevent
these processes by ubiquitinating Nek2A, Aurora-A,
Plk1, Eg5, or other key effector molecules. Whether
APC substrates other than Cdc25 are also degraded
during APCCdh1-mediated arrest remains to be seen,
but activating the APC in response to stress to directly
inhibit centrosomal processes required for chromosome
segregation is an appealing model for preserving
genomic integrity.
Chfr has recently been identified as an additional
checkpoint protein capable of delaying the G2/M
transition in response to microtubule stress (Scolnick
and Halazonetis, 2000), and new evidence indicates
that Chfr is a ubiquitin ligase that targets Plk1 for
destruction, delaying Cdc2 activation and mitotic entry
(Kang et al., 2002). The DNA damage response also
results in the inhibition of Plk1 activity in an ATR- or
ATM-dependent fashion (Smits et al., 2000; van Vugt
et al., 2001). Because Plk1 performs essential functions
in mitotic entry, centrosome maturation, spindle
function, mitotic exit, and cytokinesis, its targeted
destruction or inactivation represents an attractive
mechanism for delaying mitotic progression in response
to stress. Significantly, DNA damage not only delays
the G2/M transition, but also blocks mitotic exit in
tissue culture cells (Smits et al., 2000). In addition,
DNA damage can cause loss of mitotic centrosome
function accompanied by dissociation of g-TuRC in
early Drosophila embryos, which cycle directly between
S phase and mitosis without intervening gap phases
(Sibon et al., 2000). It will be interesting to learn
whether these mitotic blocks are accomplished via
ubiquitin-dependent proteolysis of Plk1 or other
proteins and exactly how centrosome maturation and
segregation processes are affected.
Cell cycle transitions: control by and of the centrosome
The centrosome is emerging as an important regulator
of cell cycle transitions, including the G1/S transition
(Hinchcliffe et al., 2001; Khodjakov and Rieder, 2001),
the metaphase-anaphase transition (Huang and Raff,
1999; Wakefield et al., 2000), and completion of
cytokinesis (Khodjakov and Rieder, 2001; Piel et al.,
2001). Although how the centrosome governs these
transitions is not well understood, one model is that
the centrosome acts as a central organizing center,
where enzymes and substrates are brought into close
proximity, and the signals responsible for cell cycle
progression are integrated and propagated throughout
the cell. For instance, the proteasomal machinery has
been reported to be concentrated at the centrosome
(Fabunmi et al., 2000; Wigley et al., 1999). Proteolytic
activity specifically associated with the centrosomes has
yet to be shown to directly catalyze cell cycle
transitions, but it is certainly tempting to speculate
that such localized activity is crucial to these processes.
Centrosome-mediated initiation of the metaphaseanaphase transition
Provocative evidence that centrosomes are a crucial
organizing center of ubiquitin-mediated proteolysis
comes primarily from live-cell imaging studies of
Drosophila embryos expressing GFP-cyclin B (Huang
and Raff, 1999). In these cells, GFP-cyclin B decorates
the entire mitotic spindle at metaphase. Disappearance
of GFP-cyclin B, attributed to ubiquitination by
APCCdc20, occurs in a wave that begins at the spindle
poles and proceeds to the spindle equator, after which
the chromosomes enter anaphase. This same research
group has isolated a mutation called centrosomes fall
off (cfo) with a remarkable phenotype in which the
centrosomes somehow detach from the spindle in
mitosis (Wakefield et al., 2000). In cfo mutants, GFPcyclin B disappears from the detached centrosomes but
not from the centrosome-less spindle, which arrests in
mitosis, indicating that destruction of cyclin B on the
spindle not only begins at the centrosome but also
requires an intact connection between the spindle and
the centrosome. It is also possible, however, that a
checkpoint mechanism on the spindle recognizes
centrosome detachment and prevents APC activation
and anaphase. Both papers suggest that the centrosome
acts as an organizing center for initiating proteolytic
activity at the metaphase to anaphase transition, at
least in Drosophila embryos.
Centrosome-mediated completion of cytokinesis
Accumulating evidence also implicates the centrosome
in directly triggering the completion of cytokinesis
(for review see Ou and Rattner, 2002). These studies
challenge the dogma that centriolar splitting is
uniquely associated with the onset of centriolar
duplication and that mother and daughter centrioles
maintain their orthogonal, or at least juxtaposed,
relationship from the time of the daughter centriole’s
duplication in S phase until centriolar splitting at the
following G1/S transition. Rather, it appears that
centriole position and behavior is much more
dynamic than previously thought. Live-cell imaging
of HeLa and U2OS cells stably expressing GFPcentrin as a centriolar marker demonstrated that
soon after anaphase, the centrioles in each postmitotic centrosome separate. The mother centriole
Oncogene
Ubiquitination and the centrosome cycle
DV Hansen et al
6216
maintains a fixed position in the cell center, whereas
the daughter centriole wanders throughout the
cytoplasm (Piel et al., 2000). Shortly before completion of cytokinesis (abscission), the mother centriole
transiently migrates directly to the intercellular
bridge, resulting in the release of microtubule bundles
from the midbody and narrowing of the bridge.
Abscission occurs shortly after the mother centriole
moves away and rejoins the daughter centriole in the
cell center (Piel et al., 2001).
Studies in fixed cells a decade earlier had suggested a
similar interaction of the centrosome with the intercellular bridge (Mack and Rattner, 1993). Though the
nature of the centrosome’s actual function in this
process is unknown, disassembly of microtubule
structures at the midbody could certainly involve
ubiquitin-mediated proteolysis. One possible model is
that the centrosome initiates this terminal step of cell
division through directing the activity of the APC,
which localizes to the centrosome throughout the cell
cycle (Tugendreich et al., 1995), toward relevant
substrates at the intercellular bridge. Interestingly,
misregulation of the Cdc14A phosphatase, a core
centrosomal component and activator of APCCdh1 in
human cells, causes a broad range of mitotic defects,
including cytokinesis failure (Bembenek and Yu, 2001;
Kaiser et al., 2002; Mailand et al., 2002). Of particular
interest, when Cdc14A was down-regulated by siRNA
duplexes, one of the specific defects observed was an
abortive cytokinesis in which daughter cells attempted
to separate but remained attached by the intercellular
bridge for several hours before fusing to form a
binucleate cell (Mailand et al., 2002). Although
Cdc14A was also shown in this study to have a role
in splitting the centrosome, it has been shown
elsewhere that separation of mother and daughter
centrioles is not requisite for centriole movement to
and from the intercellular bridge (Ou and Rattner,
2002), implying that the failure to undergo abscission
may be due to a specific lack of Cdc14A activity at the
intercellular bridge rather than impairment of the
mother centriole’s ability to migrate there. Other
aspects of how proteolysis controls cytokinesis have
been recently reviewed (Glotzer and Dechant, 2002).
An important regulatory system termed the mitotic
exit network (MEN) coordinates migration of the
spindle pole with APCCdh1 activation and the completion of cell division in budding yeast (see McCollum
and Gould, 2001 for review). At the top of this
signaling cascade lies the small GTPase Tem1p, which
associates with the spindle pole body (SPB), the yeast
equivalent of the centrosome. Tem1p is maintained in
its inactive, GDP-bound state until it becomes
activated upon migration of the daughter SPB into
the bud. The downstream effector of the MEN is the
Cdc14p phosphatase, which promotes downregulation
of mitotic Cdk activity through multiple mechanisms
including the activation of APCCdh1. In addition to
Tem1p, other members of the MEN also localize to the
SPB, implicating the SPB as an organizing center to
direct mitotic exit.
Oncogene
The centrosome may thus organize epigenetic
information that controls its own fate, overseeing the
same cell cycle transitions that ultimately dictate its
own duplication and segregation to daughter cells.
Therefore, various defects in centrosome function can
be expected to beget failure of the once-and-only-once
centrosome duplication restraint or defects in cell
division itself. In either case, the end result is a failure
of the cell cycle as a means of generating two daughter
cells each containing one centrosome and one complete
genome.
Proteolysis at the centrosome and cancer
While aneuploidy has long been known to be one of
the most common hallmarks of cancer, this trait has
customarily been thought to be a late event in cancer
progression resulting from the accumulation of other
genetic lesions. A growing body of evidence, however,
suggests that aneuploidy may occur at an early rather
than late stage in tumorigenesis and may even be
critical in the transition to malignancy (Lingle et al.,
2002). Because the centrosome serves as the microtubule-organizing center (MTOC) of the cell and thus
has central roles in the establishment of cytoplasmic
organization and formation of the mitotic spindle,
mutations resulting in defective or supernumerary
centrosomes have the potential to result in damage to
and/or missegregation of chromosomes through
formation of multipolar or otherwise aberrant
spindles (Brinkley, 2001; Doxsey, 1998; Duensing
and Munger, 2001; Lingle and Salisbury, 2000; Marx,
2001). We have discussed several known and
postulated mechanisms by which ubiquitin-mediated
proteolysis helps direct multiple steps in the centrosome cycle, and thus disruption of these proteolytic
events may be crucial in driving one possible
pathway toward tumorigenesis: centrosomal aberrations followed by aneuploidy and finally malignancy.
It should be noted, however, that many genetic
alterations reported to cause centrosome overduplication have not been fully characterized, and that the
phenotype of supernumerary centrosomes, or centrosome amplification, may often result more from
failures in cytokinesis rather than reinitiation of the
duplication process itself. The following paragraphs
summarize the known examples of relationships
between ubiquitin ligase misregulation, centrosomal
abnormalities and cancer.
The SCF, centrosomes and cancer
In addition to the work described above regarding the
requirement for the SCF ubiquitin ligase components
Skp1 and Cul1 for centriolar splitting at the G1/S
transition (Freed et al., 1999), other studies have
identified particular F-box proteins whose misregulation affect the centrosome duplication cycle and
genomic stability. Mice with a targeted disruption in
the F-box protein Skp2 are viable, but many animals
Ubiquitination and the centrosome cycle
DV Hansen et al
6217
show marked overduplication of their centrosomes and
extensive polyploidy (up to 12 centrosomes and 16C
DNA content) (Nakayama et al., 2000). Importantly,
however, these mice appeared healthy up to 10 months
of age and failed to display an increased incidence of
cancer, suggesting that the causal link between
centrosomal/chromosomal defects and tumorigenesis
is still not clear. Studies in Drosophila have identified
another F-box protein, Slimb/b-TrCP, that acts to
negatively regulate both Hedgehog and Wnt/Wingless
oncogenic signal transduction pathways (Jiang and
Struhl, 1998; Theodosiou et al., 1998). Defects in the
latter pathway, resulting in misregulation of b-catenin
degradation, have already been found in a significant
number of human cancers. Interestingly, Slimb has also
been implicated in limiting centrosome duplication, as
Slimb hypomorphs display increased numbers of
centrosomes as well as polyploidy (Wojcik et al.,
2000). Further investigation is required to discover
the targets of ubiquitination by these SCF complexes,
how they cause centrosome amplification, and whether
they have a role in cancer progression.
The APC, centrosomes and cancer
As previously discussed, several substrates of the APC
are also important players in the centrosome cycle. Of
these, cyclin A and kinases of the Aurora-A and Polo
families are relevant to both centrosome amplification
and cancer. First, high cyclin A/Cdk2 activity is needed
for the centrosome overduplication phenotype
observed in conditions of prolonged hydroxyurea
treatment (Balczon, 2001), and cyclin A overexpression
is an indicator of poor prognosis in breast and colon
cancers (Bukholm et al., 2001; Handa et al., 1999;
Michalides et al., 2002). Second, overexpression of
Aurora-A and Plk1 is frequently observed in various
cancers (Bischoff et al., 1998; Holtrich et al., 1994;
Sakakura et al., 2001; Tanaka et al., 1999; Tanner et
al., 2000). Homologues of both Aurora-A (Drosophila)
and Plk1 (Xenopus) have been demonstrated to regulate
microtubule dynamics in mitosis (Budde et al., 2001;
Giet et al., 2002), and the overexpression of either
Aurora-A or Plk1 can be sufficient to transform cells
and induce centrosome amplification and aneuploidy
through defects in cell division (Meraldi et al., 2002;
Smith et al., 1997; Zhou et al., 1998). Disruption of
APC function, therefore, might be expected to
contribute to tumorigenesis through stabilization of
cyclin A, Aurora-A, Plk1, and possibly other APC
substrates. The recently discovered Emi1 protein
contributes to the inactivation of APCCdh1 and
accumulation of cyclin A at the G1/S transition, and
its overexpression causes defects in mitotic progression
and cell division through APC inhibition (Hsu et al.,
2002; Reimann et al., 2001a). Moreover, Emi1 was
included in a recently published list of 231 genes,
identified by microarray profiling in a screen of 25 000
genes, whose overexpression was prognostic of a poor
clinical outcome in pre-metastatic breast cancer
patients (van’t Veer et al., 2002).
BRCA1 and p53, centrosomes and cancer
In addition to the SCF and APC ubiquitin ligases,
members of other ubiquitin ligase families are also
associated with the centrosome cycle, aneuploidy, and
cancer. BRCA1 is a well-known tumor suppressor
gene involved in hereditary cancers of the breast and
ovary (Deng and Brodie, 2000; Deng and Scott,
2000). More recently, a heterodimeric complex of
BRCA1 and BARD1 has been shown to possess
ubiquitin ligase activity through the RING domains
of each protein (reviewed in Baer and Ludwig, 2002),
although no substrates have been isolated thus far
other than the BRCA1/BARD1 heterodimer itself.
Strikingly, cancer-predisposing mutations have been
identified in the RING domain that abolish not only
BRCA1’s ubiquitin ligase activity, but also its
function in the G2/M checkpoint (Ruffner et al.,
2001). BRCA1 associates with the centrosome during
mitosis through a g-tubulin binding domain (Hsu et
al., 2001; Hsu and White, 1998). Mouse embryonic
fibroblasts (MEFs) harboring a deletion of exon 11
of BRCA1, which no longer includes the g-tubulin
binding domain, exhibit a defective G2/M checkpoint,
centrosome amplification, and aneuploidy (Brodie and
Deng, 2001; Xu et al., 1999), hinting that the mitotic
localization of BRCA1 to the centrosome is critical
for accurate cell division and genomic stability. Exon
11 also includes a nuclear localization sequence, but
NLS-deficient BRCA1 is still capable of nuclear
import and formation of DNA damage-induced
nuclear foci through association with BARD1
(Fabbro et al., 2002). Moreover, overexpression of
the BRCA1 g-tubulin binding domain causes centrosome amplification and spindle abnormalities,
presumably by preventing endogenous, full-length
BRCA1 from associating with mitotic centrosomes
(Hsu et al., 2001). Whether the ubiquitin ligase
activity of BRCA1 is required at the centrosome
for genomic stability is an important question that
remains to be addressed.
Finally, the tumor suppressor p53 also localizes to
the mitotic centrosome (Ciciarello et al., 2001; Morris
et al., 2000), and p53 loss of function results in
supernumerary centrosomes and genomic instability
(Carroll et al., 1999; Fukasawa et al., 1996; Mussman
et al., 2000; Ouyang et al., 2001; Tarapore et al., 2001).
In p53-null MEFs, multiple copies of functional
centrosomes are generated in a single cell cycle,
demonstrating that the centrosome amplification
observed in these circumstances is at least in part a
direct effect of excessive centriole duplication in S
phase (Fukasawa et al., 1996). This can be explained
by the failure of p53 to transactivate the expression of
the Cdk2 inhibitor p21. However, p53 also participates
in a G2 checkpoint (Bunz et al., 1998; Taylor and
Stark, 2001) and monitors the fidelity of mitosis
independent of its function in inhibiting centriole
duplication (Cross et al., 1995; Lanni and Jacks,
1998; Meek, 2000; Notterman et al., 1998). When cells
escape the spindle assembly checkpoint after prolonged
Oncogene
Ubiquitination and the centrosome cycle
DV Hansen et al
6218
treatment with spindle-damaging agents, they undergo
a defective cell division in which chromosomes fail to
segregate. p53 responds to this mitotic failure by
inducing a G1 arrest and preventing the tetraploid cell
from reentering the cell cycle. In the absence of p53 cell
cycle reentry is allowed, and polyploidy and centrosome amplification ensues. Indeed, p53 absence
worsened the tetraploidization and centrosome amplification phenotype observed upon overexpression of
Aurora-A or Plk1 mentioned previously (Meraldi et al.,
2002). In the presence of stress, p53 mutants can
therefore be expected to proceed through mitosis in the
presence of DNA or spindle damage, resulting in
genomic instability. Interestingly, tumors in which
centrosome amplification occurs in the presence of
wild-type p53 tend to display increased levels of
Mdm2, the E3 ubiquitin ligase responsible for degradation of p53 (Carroll et al., 1999; Setoguchi et al., 2001).
Mdm2 overexpression depletes the cell of p53, resulting
in all of the expected downstream effects including
centrosome abnormalities and chromosomal instability.
Given this fact, it seems likely that the widely quoted
frequency of mutation in the p53 gene in sporadic
cancers (greater than 50%) represents an underestimate
of the real incidence of p53 inactivation in human
cancer.
Conclusion
Currently, the number of proteins at the animal cell
centrosome is not known, but certainly hundreds of
proteins will be involved. The number of potential
ubiquitin ligases – at least based on the number of
RING finger proteins in the human genome – may also
number in the hundreds. Thus, our understanding of
ubiquitin ligase control of the centrosome is likely in its
infancy. In addition to the basic cell biology of the
centrosome, the probable connection of centrosome
biology to human disease, notably cancer, is just
beginning to be explored. As this set of centrosome
reviews attests, basic and cancer biologists will have
much to discuss about this tiny organelle in the
upcoming years.
References
Arlot-Bonnemains Y, Klotzbucher A, Giet R, Uzbekov R,
Bihan R and Prigent C. (2001). FEBS Lett., 508, 149 – 152.
Baer R and Ludwig T. (2002). Curr. Opin. Genet. Dev., 12,
86 – 91.
Balczon R, Bao L, Zimmer WE, Brown K, Zinkowski RP
and Brinkley BR. (1995). J. Cell. Biol., 130, 105 – 115.
Balczon RC. (2001). Chromosoma, 110, 381 – 392.
Bembenek J and Yu H. (2001). J. Biol. Chem., 276, 48237 –
48242.
Berdnik D and Knoblich JA. (2002). Curr. Biol., 12, 640 –
647.
Bischoff JR, Anderson L, Zhu Y, Mossie K, Ng L, Souza B,
Schryver B, Flanagan P, Clairvoyant F, Ginther C, Chan
CS, Novotny M, Slamon DJ and Plowman GD. (1998).
EMBO J., 17, 3052 – 3065.
Blangy A, Lane HA, d’Herin P, Harper M, Kress M and
Nigg EA. (1995). Cell, 83, 1159 – 1169.
Blow JJ and Hodgson B. (2002). Trends Cell. Biol., 12, 72 –
78.
Bornens M. (2002). Curr. Opin. Cell. Biol., 14, 25 – 34.
Brinkley BR. (2001). Trends Cell. Biol., 11, 18 – 21.
Brodie SG and Deng CX. (2001). Trends Genet., 17, S18 –
S22.
Budde PP, Kumagai A, Dunphy WG and Heald R. (2001). J.
Cell. Biol., 153, 149 – 158.
Bukholm IR, Bukholm G and Nesland JM. (2001). Int. J.
Cancer, 93, 283 – 287.
Bunz F, Dutriaux A, Lengauer C, Waldman T, Zhou S,
Brown JP, Sedivy JM, Kinzler KW and Vogelstein B.
(1998). Science, 282, 1497 – 1501.
Burton JL and Solomon MJ. (2000). Mol. Cell. Biol., 20,
4614 – 4625.
Carroll PE, Okuda M, Horn HF, Biddinger P, Stambrook
PJ, Gleich LL, Li YQ, Tarapore P and Fukasawa K.
(1999). Oncogene, 18, 1935 – 1944.
Castro A, Arlot-Bonnemains Y, Vigneron S, Labbe JC,
Prigent C and Lorca T. (2002). EMBO Rep., 3, 457 – 462.
Charles JF, Jaspersen SL, Tinker-Kulberg RL, Hwang L,
Szidon A and Morgan DO. (1998). Curr. Biol., 8, 497 – 507.
Oncogene
Chen F, Zhang Z, Bower J, Lu Y, Leonard SS, Ding M,
Castranova V, Piwnica-Worms H and Shi X. (2002). Proc.
Natl. Acad. Sci. USA, 99, 1990 – 1995.
Chen J and Fang G. (2001). Genes Dev., 15, 1765 – 1770.
Ciciarello M, Mangiacasale R, Casenghi M, Zaira Limongi
M, D’Angelo M, Soddu S, Lavia P and Cundari E. (2001).
J. Biol. Chem., 276, 19205 – 19213.
Cross SM, Sanchez CA, Morgan CA, Schimke MK, Ramel
S, Idzerda RL, Raskind WH and Reid BJ. (1995). Science,
267, 1353 – 1356.
Deng CX and Brodie SG. (2000). Bioessays, 22, 728 – 737.
Deng CX and Scott F. (2000). Oncogene, 19, 1059 – 1064.
Deshaies RJ. (1999). Ann. Rev. Cell. Dev. Biol., 15, 435 – 467.
do Carmo Avides M, Tavares A and Glover DM. (2001).
Nat. Cell. Biol., 3, 421 – 424.
Doxsey S. (1998). Nat. Genet., 20, 104 – 106.
Duensing S and Munger K. (2001). Biochim. Biophys. Acta,
2, M81 – M88.
Dutta A and Bell SP. (1997). Ann. Rev. Cell. Dev. Biol., 13,
293 – 332.
Fabbro M, Rodriguez JA, Baer R and Henderson BR.
(2002). J. Biol. Chem., 277, 21315 – 21324.
Fabunmi RP, Wigley WC, Thomas PJ and DeMartino GN.
(2000). J. Biol. Chem., 275, 409 – 413.
Fang G, Yu H and Kirschner MW. (1998). Mol. Cell, 2,
163 – 171.
Fisk HA and Winey M. (2001). Cell, 106, 95 – 104.
Freed E, Lacey KR, Huie P, Lyapina SA, Deshaies RJ,
Stearns T and Jackson PK. (1999). Genes Dev., 13, 2242 –
2257.
Fry AM, Descombes P, Twomey C, Bacchieri R and Nigg
EA. (2000). J. Cell. Sci., 113, 1973 – 1984.
Fry AM, Mayor T, Meraldi P, Stierhof YD, Tanaka K and
Nigg EA. (1998a). J. Cell. Biol., 141, 1563 – 1574.
Fry AM, Meraldi P and Nigg EA. (1998b). EMBO J., 17,
470 – 481.
Fukasawa K, Choi T, Kuriyama R, Rulong S and Vande
Woude GF. (1996). Science, 271, 1744 – 1747.
Ubiquitination and the centrosome cycle
DV Hansen et al
6219
Gard DL, Hafezi S, Zhang T and Doxsey SJ. (1990). J. Cell.
Biol., 110, 2033 – 2042.
Giet R, McLean D, Descamps S, Lee MJ, Raff JW, Prigent C
and Glover DM. (2002). J. Cell. Biol., 156, 437 – 451.
Giet R, Uzbekov R, Cubizolles F, Le Guellec K and Prigent
C. (1999a). J. Biol. Chem., 274, 15005 – 15013.
Giet R, Uzbekov R, Kireev I and Prigent C. (1999b). Biol.
Cell, 91, 461 – 470.
Glotzer M. (2001). Annu. Rev. Cell. Dev. Biol., 17, 351 – 386.
Glotzer M and Dechant R. (2002). Curr. Biol., 12, R344 –
R346.
Glover DM, Leibowitz MH, McLean DA and Parry H.
(1995). Cell, 81, 95 – 105.
Gmachl M, Gieffers C, Podtelejnikov AV, Mann M and
Peters JM. (2000). Proc. Natl. Acad. Sci. USA, 97, 8973 –
8978.
Gstaiger M, Marti A and Krek W. (1999). Exp. Cell. Res.,
247, 554 – 562.
Hames RS and Fry AM. (2002). Biochem. J., 361, 77 – 85.
Hames RS, Wattam SL, Yamano H, Bacchieri R and Fry
AM. (2001). EMBO J., 20, 7117 – 7127.
Handa K, Yamakawa M, Takeda H, Kimura S and
Takahashi T. (1999). Int. J. Cancer, 84, 225 – 233.
Hannak E, Kirkham M, Hyman AA and Oegema K. (2001).
J. Cell. Biol., 155, 1109 – 1116.
Hatakeyama S, Yada M, Matsumoto M, Ishida N and
Nakayama K. (2001). J. Biol. Chem., 276, 33111 – 33120.
Helps NR, Luo X, Barker HM and Cohen PT. (2000).
Biochem. J., 349, 509 – 518.
Hildebrandt ER and Hoyt MA. (2001). Mol. Biol. Cell, 12,
3402 – 3416.
Hilioti Z, Chung YS, Mochizuki Y, Hardy CF and CohenFix O. (2001). Curr. Biol., 11, 1347 – 1352.
Hinchcliffe EH, Li C, Thompson EA, Maller JL and Sluder
G. (1999). Science, 283, 851 – 854.
Hinchcliffe EH, Miller FJ, Cham M, Khodjakov A and
Sluder G. (2001). Science, 291, 1547 – 1550.
Hinchcliffe EH and Sluder G. (2001). Genes Dev., 15, 1167 –
1181.
Holtrich U, Wolf G, Brauninger A, Karn T, Bohme B,
Rubsamen-Waigmann H and Strebhardt K. (1994). Proc.
Natl. Acad. Sci. USA, 91, 1736 – 1740.
Honda R, Tanaka H and Yasuda H. (1997). FEBS Lett., 420,
25 – 27.
Hsu JY, Reimann JD, Sorensen CS, Lukas J and Jackson
PK. (2002). Nat. Cell. Biol., 4, 358 – 366.
Hsu LC, Doan TP and White RL. (2001). Cancer Res., 61,
7713 – 7718.
Hsu LC and White RL. (1998). Proc. Natl. Acad. Sci. USA,
95, 12983 – 12988.
Huang J and Raff JW. (1999). EMBO J., 18, 2184 – 2195.
Jackson PK, Eldridge AG, Freed E, Furstenthal L, Hsu JY,
Kaiser BK and Reimann JD. (2000). Trends Cell. Biol., 10,
429 – 439.
Jiang J, Ballinger CA, Wu Y, Dai Q, Cyr DM, Hohfeld J and
Patterson C. (2001). J. Biol. Chem., 276, 42938 – 42944.
Jiang J and Struhl G. (1998). Nature, 391, 493 – 496.
Kaiser BK, Zimmerman ZA, Charbonneau H and Jackson
PK. (2002). Mol. Biol. Cell, 13, 2289 – 2300.
Kamura T, Koepp DM, Conrad MN, Skowyra D, Moreland
RJ, Iliopoulos O, Lane WS, Kaelin Jr WG, Elledge SJ,
Conaway RC, Harper JW and Conaway JW. (1999).
Science, 284, 657 – 661.
Kang D, Chen J, Wong J and Fang G. (2002). J. Cell. Biol.,
156, 249 – 259.
Kashina AS, Rogers GC and Scholey JM. (1997). Biochim.
Biophys. Acta., 1357, 257 – 271.
Katayama H, Zhou H, Li Q, Tatsuka M and Sen S. (2001). J.
Biol. Chem., 276, 46219 – 46224.
Khodjakov A and Rieder CL. (1999). J. Cell. Biol., 146,
585 – 596.
Khodjakov A and Rieder CL. (2001). J. Cell. Biol., 153,
237 – 242.
Kramer ER, Scheuringer N, Podtelejnikov AV, Mann M and
Peters JM. (2000). Mol. Biol. Cell, 11, 1555 – 1569.
Kwon YG, Lee SY, Choi Y, Greengard P and Nairn AC.
(1997). Proc. Natl. Acad. Sci. USA, 94, 2168 – 2173.
Lacey KR, Jackson PK and Stearns T. (1999). Proc. Natl.
Acad. Sci. USA, 96, 2817 – 2822.
Lane HA and Nigg EA. (1996). J. Cell. Biol., 135, 1701 –
1713.
Lanni JS and Jacks T. (1998). Mol. Cell. Biol., 18, 1055 –
1064.
Lingle WL, Barrett SL, Negron VC, D’Assoro AB, Boeneman K, Liu W, Whitehead CM, Reynolds C and Salisbury
JL. (2002). Proc. Natl. Acad. Sci. USA, 99, 1978 – 1983.
Lingle WL and Salisbury JL. (2000). Curr. Top. Dev. Biol.,
49, 313 – 329.
Lu Z, Xu S, Joazeiro CA, Cobb MH and Hunter T. (2002).
Mol. Cell, 9, 945 – 956.
Mack G and Rattner JB. (1993). Cell. Motil. Cytoskeleton,
26, 239 – 247.
Mailand N, Lukas C, Kaiser BK, Jackson PK, Bartek J and
Lukas J. (2002). Nat. Cell. Biol., 4, 318 – 322.
Marshall WF. (2001). Curr. Biol., 11, R487 – R496.
Marx J. (2001). Science, 292, 426 – 429.
Matsumoto Y, Hayashi K and Nishida E. (1999). Curr. Biol.,
9, 429 – 432.
Mayor T, Stierhof YD, Tanaka K, Fry AM and Nigg EA.
(2000). J. Cell. Biol., 151, 837 – 846.
McCollum D and Gould KL. (2001). Trends Cell. Biol., 11,
89 – 95.
Meek DW. (2000). Pathol. Biol. (Paris), 48, 246 – 254.
Meraldi P, Honda R and Nigg EA. (2002). EMBO J., 21,
483 – 492.
Meraldi P, Lukas J, Fry AM, Bartek J and Nigg EA. (1999).
Nature Cell. Biol., 1, 88 – 93.
Meselson M and Stahl FW. (1958). PNAS, 44, 671.
Michalides R, van Tinteren H, Balkenende A, Vermorken
JB, Benraadt J, Huldij J and van Diest P. (2002). Br. J.
Cancer, 86, 402 – 408.
Morris VB, Brammall J, Noble J and Reddel R. (2000). Exp.
Cell. Res., 256, 122 – 130.
Mussman JG, Horn HF, Carroll PE, Okuda M, Tarapore P,
Donehower LA and Fukasawa K. (2000). Oncogene, 19,
1635 – 1646.
Nakayama K, Nagahama H, Minamishima YA, Matsumoto
M, Nakamichi I, Kitagawa K, Shirane M, Tsunematsu R,
Tsukiyama T, Ishida N, Kitagawa M and Hatakeyama S.
(2000). EMBO J., 19, 2069 – 2081.
Noton E and Diffley JF. (2000). Mol. Cell, 5, 85 – 95.
Notterman D, Young S, Wainger B and Levine AJ. (1998).
Oncogene, 17, 2743 – 2751.
O’Connell MJ, Walworth NC and Carr AM. (2000). Trends
Cell. Biol., 10, 296 – 303.
Ohta T, Michel JJ, Schottelius AJ and Xiong Y. (1999). Mol.
Cell, 3, 535 – 541.
Okuda M, Horn HF, Tarapore P, Tokuyama Y, Smulian
AG, Chan PK, Knudsen ES, Hofmann IA, Snyder JD,
Bove KE and Fukasawa K. (2000). Cell, 103, 127 – 140.
Ou YY and Rattner JB. (2002). Cell. Motil. Cytoskeleton, 51,
123 – 132.
Oncogene
Ubiquitination and the centrosome cycle
DV Hansen et al
6220
Ouyang X, Wang X, Xu K, Jin DY, Cheung AL, Tsao SW
and Wong YC. (2001). Biochim. Biophys. Acta., 1541,
212 – 220.
Paintrand M, Moudjou M, Delacroix H and Bornens M.
(1992). J. Struct. Biol., 108, 107 – 128.
Patterson C. (2002). Science’s STKE [online resource]: http://
stke.sciencemag.org/cgi/content/full/OC_sigtrans; 2002/
116/pe4.
Pfleger CM and Kirschner MW. (2000). Genes Dev., 14, 655 –
665.
Pfleger CM, Lee E and Kirschner MW. (2001a). Genes Dev.,
15, 2396 – 2407.
Pfleger CM, Salic A, Lee E and Kirschner MW. (2001b).
Genes Dev., 15, 1759 – 1764.
Piel M, Meyer P, Khodjakov A, Rieder CL and Bornens M.
(2000). J. Cell. Biol., 149, 317 – 330.
Piel M, Nordberg J, Euteneuer U and Bornens M. (2001).
Science, 291, 1550 – 1553.
Rao PN and Johnson RT. (1970). Nature, 225, 159 – 164.
Reimann JD, Freed E, Hsu JY, Kramer ER, Peters JM and
Jackson PK. (2001a). Cell, 105, 645 – 655.
Reimann JD, Gardner BE, Margottin-Goguet F and Jackson
PK. (2001b). Genes Dev., 15, 3278 – 3285.
Roghi C, Giet R, Uzbekov R, Morin N, Chartrain I, Le
Guellec R, Couturier A, Doree M, Philippe M and Prigent
C. (1998). J. Cell. Sci., 111, 557 – 572.
Ruffner H, Joazeiro CA, Hemmati D, Hunter T and Verma
IM. (2001). Proc. Natl. Acad. Sci. USA, 98, 5134 – 5139.
Sakakura C, Hagiwara A, Yasuoka R, Fujita Y, Nakanishi
M, Masuda K, Shimomura K, Nakamura Y, Inazawa J,
Abe T and Yamagishi H. (2001). Br. J. Cancer, 84, 824 –
831.
Sawin KE, LeGuellec K, Philippe M and Mitchison TJ.
(1992). Nature, 359, 540 – 543.
Sawin KE and Mitchison TJ. (1995). Proc. Natl. Acad. Sci.
USA, 92, 4289 – 4293.
Schwab M, Neutzner M, Mocker D and Seufert W. (2001).
EMBO J., 20, 5165 – 5175.
Scolnick DM and Halazonetis TD. (2000). Nature, 406, 430 –
435.
Setoguchi A, Okuda M, Nishida E, Yazawa M, Ishizaka T,
Hong SH, Hisasue M, Nishimura R, Sasaki N, Yoshikawa
Y, Masuda K, Ohno K and Tsujimoto H. (2001). Am. J.
Vet. Res., 62, 1134 – 1141.
Shah JV and Cleveland DW. (2000). Cell, 103, 997 – 1000.
Shirayama M, Zachariae W, Ciosk R and Nasmyth K.
(1998). EMBO J., 17, 1336 – 1349.
Sibon OC, Kelkar A, Lemstra W and Theurkauf WE. (2000).
Nat. Cell. Biol., 2, 90 – 95.
Skowyra D, Koepp DM, Kamura T, Conrad MN, Conaway
RC, Conaway JW, Elledge SJ and Harper JW. (1999).
Science, 284, 662 – 665.
Oncogene
Smith MR, Wilson ML, Hamanaka R, Chase D, Kung H,
Longo DL and Ferris DK. (1997). Biochem. Biophys. Res.
Commun., 234, 397 – 405.
Smits VA, Klompmaker R, Arnaud L, Rijksen G, Nigg EA
and Medema RH. (2000). Nat. Cell. Biol., 2, 672 – 676.
Sudo T, Ota Y, Kotani S, Nakao M, Takami Y, Takeda S
and Saya H. (2001). EMBO J., 20, 6499 – 6508.
Taguchi S, Honda K, Sugiura K, Yamaguchi A, Furukawa K
and Urano T. (2002). FEBS Lett., 519, 59 – 65.
Tan P, Fuchs SY, Chen A, Wu K, Gomez C, Ronai Z and
Pan ZQ. (1999). Mol. Cell, 3, 527 – 533.
Tanaka T, Kimura M, Matsunaga K, Fukada D, Mori H and
Okano Y. (1999). Cancer Res., 59, 2041 – 2044.
Tang Z, Li B, Bharadwaj R, Zhu H, Ozkan E, Hakala K,
Deisenhofer J and Yu H. (2001). Mol. Biol. Cell, 12, 3839 –
3851.
Tanner MM, Grenman S, Koul A, Johannsson O, Meltzer P,
Pejovic T, Borg A and Isola JJ. (2000). Clin. Cancer Res., 6,
1833 – 1839.
Tarapore P, Horn HF, Tokuyama Y and Fukasawa K.
(2001). Oncogene, 20, 3173 – 3184.
Taylor WR and Stark GR. (2001). Oncogene, 20, 1803 – 1815.
Theodosiou NA, Zhang S, Wang WY and Xu T. (1998).
Development, 125, 3411 – 3416.
Tugendreich S, Tomkiel J, Earnshaw W and Hieter P. (1995).
Cell, 81, 261 – 268.
Uto K and Sagata N. (2000). EMBO J., 19, 1816 – 1826.
van’t Veer LJ, Dai H, van de Vijver MJ, He YD, Hart AA,
Mao M, Peterse HL, van der Kooy K, Marton MJ,
Witteveen AT, Schreiber GJ, Kerkhoven RM, Roberts C,
Linsley PS, Bernards R and Friend SH. (2002). Nature,
415, 530 – 536.
van Vugt MA, Smits VA, Klompmaker R and Medema RH.
(2001). J. Biol. Chem., 276, 41656 – 41660.
Vidwans SJ, Wong ML and OFarrell PH. (1999). J. Cell.
Biol., 147, 1371 – 1378.
Wakefield JG, Huang JY and Raff JW. (2000). Curr. Biol.,
10, 1367 – 1370.
Wigley WC, Fabunmi RP, Lee MG, Marino CR, Muallem S,
DeMartino GN and Thomas PJ. (1999). J. Cell. Biol., 145,
481 – 490.
Wojcik EJ, Glover DM and Hays TS. (2000). Curr. Biol., 10,
1131 – 1134.
Xu X, Weaver Z, Linke SP, Li C, Gotay J, Wang XW, Harris
CC, Ried T and Deng CX. (1999). Mol. Cell, 3, 389 – 395.
Zheng N, Schulman BA, Song L, Miller JJ, Jeffrey PD,
Wang P, Chu C, Koepp DM, Elledge SJ, Pagano M,
Conaway RC, Conaway JW, Harper JW and Pavletich NP.
(2002). Nature, 416, 703 – 709.
Zhou H, Kuang J, Zhong L, Kuo WL, Gray JW, Sahin A,
Brinkley BR and Sen S. (1998). Nat. Genet., 20, 189 – 193.
Ubiquitination and the centrosome cycle
DV Hansen et al
6221
Appendix – A primer on ubiquitylation enzymes
E3, a ubiquitin ligase
Ubiquitin-dependent proteolysis occurs following the
covalent addition of a polyubiquitin chain to a specific
target protein. Polyubiquitylation targets proteins to
the 26S proteasome, an ATP- and ubiquitin-dependent
protease complex. The cascade of E1, E2 and E3
enzymes activate the ubiquitin and facilitate the
assembly of the multiubiquitin chain. Generally, the
E3s are the most diverse group.
The E3 ubiquitin ligases couple with the E2s to
bind to substrate and assemble a multiubiquitin
chain on the substrate. For the HECT domain E3s,
the E3 itself forms a thioester with ubiquitin and
presumably participates in transferring the ubiquitin
directly to the substrate. For the E3 complexes
containing cullins or RING finger proteins, no
direct thioester between E3 and ubiquitin has been
identified. In these E3 classes, the ubiquitin ligase
might function to facilitate the interaction between
substrate and E2 enzyme (see main text for further
discussion).
Ubiquitin (Ub)
A 76 amino acid protein with the C-terminal glycine
(Gly76) residue capable of forming an isopeptide
bond with a side chain lysine on a target protein or
one of the side chain lysines of ubiquitin itself
(notably lysine 48). There are a number of ubiquitinlike molecules (UBLs), such as SUMO-1 and
NEDD8/Rub1, that are added as monomers to
side-chain lysines.
E4, a multiubiquitin chain assembly factor
The budding yeast UFD2 has been called an E4
protein. The protein binds to multiubiquitin chains and
may facilitate part of the ubiquitin-dependent proteolysis pathway following ubiquitin chain assembly.
E1, a ubiquitin-activating enzyme
The 26S proteasome
Ubiquitin is activated by the E1 enzyme and ATP to
form a thioester linkage between the C-terminal
glycine 76 and a conserved active site cysteine.
Uba1 is the major form of this enzyme in yeast
and humans.
The proteasome is an abundant protease complex
comprising a 20S core particle (CP) flanked by two 19S
regulatory particles (RP). The CP is cylindrical, with
narrow channels feeding a central cavity with multiple
protease sites. The regulatory particles regulate
substrate access on both ends of the core. The RP
itself contains a base, which contains ATPase subunits
thought to unfold substrates for access to the core, and
a lid, which contains subunits for binding multiubiquitin chains and ubiquitin-deconjugating enzymes
(isopeptidases).
E2, a ubiquitin-conjugating enzyme
After activation by the E1, ubiquitin is transesterified
to a conserved cysteine of an E2 enzyme. There are
13 E2s in yeast and approximately 30 – 50 in
vertebrates. The genetic name for these enzymes
includes the three letter code ‘Ubc’. Except for
Ubc9 (a SUMO-conjugating enzyme) and Ubc12 (a
NEDD8/Rub1 conjugating enzyme), the Ubcs have
varying genetically defined functions in ubiquitylation,
but some overlapping roles. Ubc3 is also known as
Cdc34, a crucial E2 enzyme in the SCF ubiquitin
ligase.
Ubiquitin hydrolases (isopeptidases)
A diverse group of enzymes that hydrolyse isopeptide
bonds in a multiubiquitin chain. These enzymes might
provide regulatory and proofreading functions for
assembly of ubiquitin chains and also allow recycling
of ubiquitin monomers.
Oncogene